Aspirin is one of the most widely used drugs in the world. Regular use is associated with reduced risk of mortality in all cardiovascular risk groups. It is an anti-inflammatory, analgesic and anti-pyretic agent and is used in the fight against cardiovascular disease and is predicted to have a role in preventing colorectal, oesophageal, gastric and lung cancers (e.g. Chan 2005) as well as stroke, Alzheimer's disease (Etminan et al., 2003) and other forms of dementia. Some models predict that daily aspirin consumption by people over fifty years would double their chances of living until their 90's (Morgan, 2003).
The main side effects associated with aspirin use are gastrointestinal. Aspirin causes dyspepsia in nearly half of all patients and it triples the risk of GI bleeding. Endoscopically controlled studies demonstrate an increased risk of bleeding at all aspirin doses even at the relatively low doses used in the prevention of myocardial infarction (MI). In one study, 10% of patients on low dose aspirin (10-300 mg/day) had endoscopic ulcers after 12 weeks, with one case occurring at 10 mg/day (Cryer & Feldman, 1999, Cryer 2002). Several studies have shown bleeding commences 5 to 30 days after the start of therapy indicating that adaptation does not occur. Significantly, the risk of GI side-effects has limited aspirin use to patient groups with a high probability of a thrombotic event: in a random population the risk of serious GI injury is higher than the risk of aspirin-preventable death. As of yet, there are no reliable dose-related data for the prophylactic use of aspirin in cancer, however, it seems likely to be higher than the optimal dose required for its established role in the prevention of heart attack and so there is likely to be an increased risk of greater GI toxicity. Although the absolute risks are low (1-2%), its widespread and rapidly growing consumption causes aspirin-induced gastrointestinal toxicity to be a public health concern (Morgan, 2003; Laheij 2001; Newton et al., 2004). A number of contributions to aspirin GI toxicity are recognised. The gut wall is protected from the harsh luminal contents by a protective layer. This barrier is partly maintained by the two cyclooxygenase enzymes (COX-1 & COX-2). The cardiovascular protective effects of aspirin stem from its inhibition of platelet cyclooxygenase enzyme COX-1, while its cytoprotective effects have been attributed to its unique ability to acetylate the cyclooxygenase enzyme COX-2 which causes arachidonic acid to be shunted away from PGE2, a cancer promoter, towards HETE, a cancer suppressor. COX-2 also has role in wound healing in the GIT. Aspirin inhibits these enzymes as it passes through the gut during absorption and so attenuates their protective role. The biochemical aspect of toxicity therefore results from local inhibition of COX-1 and COX-2 by aspirin which leads to suppression of the prostaglandins (PGE2, PGI2) that normally regulate gastric acid secretion and blood flow. There is also a clear chemical aspect to aspirin's toxicity. Aspirin is a hydrophobic acid (pKa 3.5). It is lipid soluble at low pH values and it is able to disrupt the hydrophobic layer covering the epithelium allowing access by luminal contents, causing irritation, eventually leading to ulceration. This may be a more important cause of toxicity than the biochemical component. In one recent study, the administration of oral aspirin to rats caused gastric lesions whereas there was no gastric damage when the drug was given subcutaneously, despite evidence of inhibition of COX from both routes (Mahita, 2006). Drug-induced GI toxicity is very complex and the subject of frequently conflicting findings but this particular study indicates that chemical toxicity is significant.
The problem of GI toxicity has been the focus of pharmaceutical attention for many years, but endoscopic studies demonstrate that conventional solutions such as enteric coatings or buffering are at best inadequate (Kelly et al, 1996, Walker et al., 2007). Thus establishing new ways of delivering aspirin or dealing with its GI effects is a matter of public health importance and a significant commercial opportunity.
A potentially valuable solution to the problem is the design of aspirin derivatives capable of delaying aspirin release from occurring in the GI tract until after absorption into plasma. Such a derivative should properly be termed a prodrug. Prodrugs are therapeutic agents that are themselves inactive but on metabolism form active agents (Albert, 1958). Aspirin prodrugs were investigated for many years as a means of depressing its gastric toxicity (Jones, 1985).
The original aspirin prodrug rationale proposed that blocking the aspirin carboxylic acid, for example with an ester, would effectively abolish the chemical aspect of gastric toxicity which results from direct contact between the aspirin carboxylic acid and the gastric mucosa. Aspirin esters that are activated during the passage through the gastrointestinal epithelium are expected to exhibit greatly reduced gastric toxicity if this model is correct, even if drug release happens within the epithelial cells.
When the biochemical component of aspirin toxicity became more widely appreciated, the prodrug rationale was refined. In contrast to aspirin, its esters do not have the ability to inhibit COX. They would therefore not interrupt the synthesis of protective prostaglandins during passage through the gut wall. Then, following absorption, esterases in the blood would break the ester, releasing aspirin. The drug would later reach the gut through the systemic circulation but at a much lower concentration; aspirin is rapidly metabolised in the body and has a half-life of only 20 minutes. It is suspected that effective blockade of the COX-dependent mucosal defence systems requires a rather high concentration of aspirin. This is because aspirin is a weak inhibitor of one of the COX enzymes (COX-1) and a very weak inhibitor of the other (COX-2). In other words, aspirin inhibits both of the protective enzymes during the absorption phase but it is unlikely to achieve the required concentration to block both enzymes after distribution throughout the body following absorption (a similar kind of pharmacokinetic argument explains aspirin's selective inhibition of platelet thromboxane A2 over endothelial prostacyclin in the heart (Pedersen A. K. & FitzGerald G. A. 1984)).
Aspirin prodrug esters are therefore expected to have lower GI toxicity because they would not cause topical irritancy, the first passage of aspirin through GI at high concentration would be avoided and the second distribution is likely to be at a concentration that would leave COX-2 dependent protective functions intact. The idea of such aspirin ester prodrugs is attractive since the prodrugs are not acidic during passage across the protective barrier and would not disrupt it. They are also expected to have a smaller impact on the biochemical machinery regulating the barrier, whether activated in the epithelium or especially later after entry into the blood stream. This has the advantage that aspirin associated adverse GI effects are avoided. The drug is safer since it is not activated until after it has passed through the GI tract (FIG. 1).
Another problem with aspirin from a clinical point of view is that it is unstable towards moisture and can't therefore be formulated in solutions. Aqueous solutions of aspirin would be especially desirable in paediatric and geriatric medicine. One of the major contributors to aspirin's instability is a form of autocatalysis first described by Jencks and Pierre (1958). Aspirin has a carboxylic acid group and an acetyl group. The carboxylic acid group has the capacity to activate a nearby water molecule generating hydroxide which attacks the acetyl group. By forming an ester of aspirin the carboxylic acid group is masked and cannot engage in autocatalysis. Aspirin esters are usually more stable than aspirin and therefore have the potential to be formulated in a variety of useful ways that can't be applied to aspirin. This second advantage to aspirin esters has been well established experimentally.
On the other hand, the hypothesized theory of obviation of aspirin toxicity has never been tested because there has never been a suitable aspirin ester prodrug candidate. This is because aspirin ester prodrugs are very difficult to design. An aspirin ester of paracetamol—benorylate was on the market for about thirty years until it emerged that its administration dose to humans does not result in aspirin release (Williams et al., 1989).
The problem with aspirin esters and related derivatives is a metabolic one. Aspirin esters are converted in the body to salicylic acid rather than aspirin (Nielsen & Bundgaard, 1989). Aspirin esters are metabolised in human tissue and blood by way of the possible pathways shown in FIG. 2. An effective aspirin prodrug should be cleaved at the carrier group liberating aspirin following absorption. Rapid hydrolysis of aspirin esters happens in blood and plasma (t1/2<1 min), but not at the desired aspirin-carrier ester bond (position B in FIG. 2). Instead, the acetyl group is cleaved (A in FIG. 2) and the resultant product is a salicylate ester, and ultimately salicylic acid. This biochemical pathway cannot produce aspirin. The ratio of salicylate to aspirin is usually greater than 99:1, regardless of the identity of the carrier group (the alcohol component of the prodrug used to form the ester with the drug carboxylic acid is known as the carrier in prodrug terminology). If you form an ester from an acid drug the part you are attaching blocks the acid chemistry but it also confers on the new entity some of its own physicochemical characteristics (et FIG. 2). This problem has elicited interest both as a pharmaceutical conundrum and a commercial opportunity. There is a substantial body of academic and patent literature in the area (See Gilmer et al., 2002 and references therein). However the vast majority of compounds referred to as aspirin prodrugs in the literature do not actually function as aspirin prodrugs in vitro or in vivo and they release the corresponding salicylate ester instead (Nielsen & Bundgaard, 1989).
In order for an aspirin ester to function as a prodrug hydrolysis in blood cleavage has to occur at the carrier ester bond. The design challenge is that esterifying aspirin causes the wrong ester group to undergo hydrolysis in the presence of human plasma. The problem was first explained by Bungaard and Nielsen (1989). When aspirin enters the blood stream its acetyl group is hydrolysed by the dominant esterase enzyme in human plasma-butyrylcholinesterase (BuChE), resulting in the formation of salicylic acid. Aspirin is negatively charged at blood pH and butyrylcholinesterase is not actually at its most efficient when processing negatively charged substrates. By esterifying aspirin the negative charge (which suppresses metabolism) is removed and the acetyl group becomes a much better substrate for butyrylcholinsterase. Introduction of the new ester group therefore greatly accelerates the rate of metabolism of the existing acetyl ester. For example, aspirin has a half-life of around one hour in dilute plasma but aspirin esters undergo the same deacetylation process with a half-life of less than one minute: neutral phenylacetates, such as aspirin esters are among the most efficiently hydrolysed substrate types of butyrylcholinesterase. An interpretation of this in terms of basic enzymology is that the aspirin ester fits the enzyme better than aspirin itself. Bundgaard recognised that in order for metabolism to occur at the correct point, the carrier group has to have a structure of competing complementarity to the acetyl group i.e. it has to be at least as attractive a substrate for the BuChE enzyme as the acetyl group. Even better carrier groups can be envisaged that promote their own hydrolysis while at the same time suppressing hydrolysis of the neighbouring acetyl group. The butyrylcholinesterase enzyme takes its name from its efficiency in hydrolysing esters of choline. Neilsen and Bungaard studied glycolamide esters of aspirin where the carrier group was designed to mimic choline so that its detachment might successfully compete with acetyl group hydrolysis. The glycolamides were only partially successful with the most successful example being hydrolysed around 50% in both the desirable and unproductive directions (routes A and B in FIG. 2). Nielsen and Bundgaard's work established the important principle that a successful aspirin prodrug requires a carrier group that fits human plasma esterase in a manner that overrides its preference for the acetyl group. This turns out to be a highly demanding requirement to which their response was only partly adequate. However, apart from the technology described herein, the glycolamides are the only known compounds which can even partly be described as true aspirin prodrugs.
Another strategy that has been adopted from time to time is to design esters where the aspirin-carrier bond is so labile that it breaks before esterases can attack the acetyl group. The problem with this approach is that aspirin is already quite unstable towards hydrolysis by water and other nucleophiles at its acetyl group. Introducing a second chemically active ester has the effect of heightening the reactivity of the acetyl group (as well as adding another point of lability). Aspirin esters deliberately intended to undergo cleavage by chemical stimuli such as water therefore have the obvious flaw that they are likely to encounter such stimuli during storage and are therefore susceptible to degradation on the shelf. This negates one of the advantages of aspirin ester prodrugs in the first place—that they are more stable than aspirin towards moisture. Prodrugs designed to be cleaved in response to generic chemical stimuli tend also to break under conditions found in the GIT, which they would meet before absorption.
Interest in the aspirin prodrug area has intensified with the advent of the so-called nitric oxide (NO)-aspirins, which are a type of aspirin ester but with an NO.-releasing moiety attached to the carrier group. The main rationale for the development of NO-aspirins is that NO. promotes mucosal defence, offsetting the damage caused by aspirin (Fiorucci and Del Soldato, 2003). This concept is now well accepted in the biomedical community. Nitric oxide and aspirin also have complementary and sometimes synergistic pharmacological effects so the combination is expected to show a greater range of pharmacological effects than aspirin alone. NO release protects the stomach from aspirin induced gastric erosion by promoting blood flow and reducing leucocyte adhesion whiles its antithrombotic properties through the GMP pathway potentiate the antiplatelet effects arising from COX-1 inhibition by aspirin. It is thus considered reasonable to link them as an ester in an attempt to produce a mutual prodrug of aspirin and nitric oxide. NCX-4016 (NicOx SA, France) is a prototype compound for NO-aspirin drugs (WO 95/030641, WO 97/16405, WO97/16405, WO0044705). It produces NO in vivo and has anti-platelet effects. NCX-4016 exhibits greater gastric tolerability than aspirin in several animal modes. NCX-4016 began preclinical development in 1996 and since 2002 has been evaluated in the treatment of cardiovascular disorders (e.g. Peripheral Arterial Occlusive Disease (PAOD) (Phase II)), colon cancer prophylaxis (Phase I) and cancer pain.
NCX-4016 was one of the most widely touted pharmaceutical developments of the past decade and was regarded as a significant biomedical advance (see for example Levin, 2004). However, as an NO.-aspirin prodrug, NCX-4016 appears to have a significant design flaw. The key test for an aspirin ester prodrug is whether it hydrolysed to aspirin or its salicylate ester when it is incubated in human plasma or blood. NCX-4016 is an aspirin ester of a substituted phenol. There are no published data on the hydrolysis pattern of NCX-4016 in human plasma but there are for similar esters—the aspirin ester of paracetamol (benorylate—Williams et al, 1989), the aspirin ester of guicaol (Qu et al., 1990), and the aspirin ester of phenol (Nielsen & Bundgaard, 1989; also see Table 8). None of these compounds produced more than 0.5% aspirin when incubated in relevant biological matrices. There is therefore no direct evidence that NCX4016 can or should produce aspirin. In vivo and in vivo metabolic studies on the compound refer only to salicylate metabolites (Carini et al., 2002). Furthermore, COX inhibition with NCX-4016 is less extensive than with aspirin. This is a significant deficiency because platelet COX-inhibition needs to be quite complete to prevent human platelet aggregation. Another recent study suggests that NCX-4016 may act directly on its target without the release of aspirin (Corazzi et al., 2005). There have been a number of other recent efforts to design compounds capable of liberating both aspirin and nitric oxide in human tissue. The results have been disappointing. All reported compounds undergo hydrolysis along the typical salicylate pathway and fail to liberate significant amounts of aspirin though they are potentially capable of releasing nitric oxide (Gilmer et al., 2007; Valezquez et al., 2005; Cena et al., 2003).
International Publication No. WO9403421 describes salicylate esters of the clinically used isosorbide nitrate, ISMN. The compound described is isosorbide-mono-nitrate aspirinate (ISMNA) and its potential use in a transdermal patch is discussed.

The compound was said to be useful for its antianginal and platelet washing properties. Chemical hydrolysis studies were reported to show degradation with production of isosorbide-mono-nitrate (ISMN), salicylic acid and aspirin which expressed platelet washing and anti-angina activities. However, it was not expected that ISMNA could act as a viable aspirin prodrug because no other aspirin ester had been shown to act as an aspirin prodrug, apart from the glycolamides, and these were very deliberately designed to be complementary to plasma BuChE. However ISMNA turned out to be a potent inhibitor of platelet aggregation in rabbit tissue in vitro and it was later shown that ISMNA is efficiently converted to aspirin by rabbit plasma esterases. It was tested in an oral study in dogs in which it was compared with aspirin in two of aspirin's pharmacological hallmarks: inhibition of the biosynthesis of thromboxane (a biochemical that stimulates platelets to aggregate) and functional inhibition of platelet aggregation. ISMNA showed weak effects on both markers indicating that it released only small amounts of aspirin in the dog. By incubating ISMNA in dog blood and monitoring its hydrolysis we were able to show that it is not converted effectively to aspirin by dog esterases because of differences between the esterases in dog and rabbit blood. Later it emerged that ISMNA is not hydrolysed productively in human plasma either. In human plasma solution and in human blood in vitro, ISMNA produces >90% salicylate and <10% aspirin. ISMNA is correspondingly much less potent than aspirin as an inhibitor of platelet aggregation in human whole blood and human platelet rich plasma (its IC50 is 85 μM compared with 5 μM for aspirin in human platelet aggregation to arachidonic acid in platelet rich plasma). The results taught that for an ester of aspirin, ability to inhibit platelet aggregation or thromboxane synthesis correlates with ability to produce aspirin: an inefficient prodrug makes for an ineffective inhibitor of platelet aggregation. The low level of aspirin release and lack of potency precluded isosorbide-mono-nitrate aspirinate (ISMNA) from being a viable drug candidate for humans.
International Publication No. WO9817673 discloses the di-aspirinate of isosorbide and two mono-aspirinate esters of isosorbide, namely isosorbide-2-aspirinate and isosorbide-5-aspirinate. Isosorbide-di-aspirinate (ISDA), the principal subject of WO9817673, was not ostensibly any different from the many other earlier ester prodrug candidates that have been tested.

Moreover, the person skilled in the art would not have expected Isosorbide-di-aspirinate (ISDA) to function as a viable aspirin prodrug and would have had no chemical or biochemical reason to believe that hydrolysis would lead to anything other than acetyl group cleavage and so ultimately salicylic acid. It was very surprising therefore when we were able to show in our own laboratory that ISDA inhibits platelet aggregation in rabbit platelet rich plasma. It also had an inhibitory effect on thromboxane synthesis following oral administration to a group of dogs (Gilmer et al., 2003). The aspirin-like properties indicated that the hydrolysis of ISDA in plasma leads to some aspirin. ISDA was shown to undergo rapid hydrolysis when incubated in phosphate buffered human plasma solutions to produce approximately 60% aspirin (Gilmer et al., 2002). The remaining 40% of the compound was hydrolysed along the unproductive salicylate pathway. The study indicated that a specific enzyme present in human plasma catalyses aspirin release from isosorbide diaspirinate (ISDA). It was confirmed that butyrylcholinesterase was the human plasma enzyme involved. Closely related horse plasma butyrylcholinesterase generated only 11% aspirin. Gilmer et al (2001, 2002) further describe the hydrolysis characteristics and biological effects of isosorbide-mono-nitrate aspirinate (ISMNA) and isosorbide-di-aspirinate (ISDA).
The diaspirinate ester ISDA and the glycolamide esters of Nielsen and Bungaard are the only esters in the chemical literature that can to a significant extent act as aspirin prodrugs in human plasma. Compounds not producing aspirin as a hydrolysis product are better classified as salicylic acid prodrugs. For example, in the present context, ISMNA is an aspirin prodrug only in rabbit tissue but it is a salicylic acid prodrug in human blood.
There is a pressing need for better aspirin prodrug compounds because of their intrinsic therapeutic potential and because of the demand for compounds capable of releasing both aspirin and nitric oxide. A nitric-oxide releasing aspirin ester must in the first instance be an ester capable of undergoing conversion to aspirin in the key plasma hydrolysis model. In particular it is desirable to provide aspirin prodrug compounds which resist aqueous hydrolysis and α-chymotrypsin, yet will undergo rapid hydrolysis in the presence of human plasma to liberate aspirin and potentially other pharmacologically active moieties, in particular nitric oxide.